Cell-cell interactions during apoptosis of cancer cells
The aim of the research project is to study the interaction between apoptotic and healthy cancer cells, with particular focus on the bystander effect and tunnelling nanotube (TNT)-mediated cell-to-cell communication. In last four years, we have obtained significant scientific achievements and fulfilled the majority of specific objectives set in the grant proposal. 1. Spatio-temporal analysis of bystander effects Tamoxifen is regarded as the most successful chemotherapeutical agent used for the management of breast cancer. However, it is not clear if tamoxifen can induce bystander killing of non-treated cancer cells and the dynamics of this response. Therefore, we studied the bystander response between tamoxifen-treated and non-treated breast cancer cells (MCF-7 cells) in a microfluidic device to ensure selective drug delivery. Our data show that tamoxifen does induce a bystander response, which is detectable as early as one hour after drug treatment and reached effective distances of at least 2.8 mm. Furthermore, the bystander effect was merely dependent on diffusible factors rather than cell contact-dependent signalling. 2. Structural and functional analysis of TNTs in apoptotic cancer cells. (1) The role of apoptotic regulators in TNT formation p53 is an important tumour suppressor protein that regulates apoptosis and was suggested to be essential for the formation of tunnelling nanotubes (TNTs) in astrocytes. We investigated whether p53 is a key protein for the formation of TNTs. Our data show that activation of p53 does not promote TNT formation in p53WT PC12 cells. In addition, p53-negative SAOS-2 and MSCs can form TNTs and exogeneous expression of p53 does not induce TNTs in SAOS-2 cells. We concluded that the existence of TNTs appears to be more connected to cell type and not to the existence or activation of p53. (2) Formation and function of TNTs in cells under apoptotic stress In this study, we found that PC12 cells treated with ultraviolet light (UV) were rescued when co-cultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterised by continuous microtubule localized inside these TNTs. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c, but did not enter into the execution phase of apoptosis characterised by caspase-3 activation. Moreover, mitochondria co-localised with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the co-culture. We conclude that TNTs-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. (3) Role of PS-positive TNT-connections in phagocytosis The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells promotes phagocytosis by phagocytes. We observed that apoptotic PC12 cells connected with neighbouring untreated cells via TNTs composing PS-exposed membrane and facilitating the transfer of PS-exposed membrane from apoptotic to live cells. In addition, other signals attracting phagocytes, such as oxidized phospholipids and calreticulin, were also transferred to live cells. In addition, anti-phagocytic signal CD47 presenting on live cell membrane was masked by the transferred membrane. Confocal imaging revealed an increase of phagocytosis of live PC12 cells by murine RAW264.7 macrophages when the live PC12 cells were cocultured with UV-treated PC12 cells. The phagocytosis of live cells was inhibited by 50 nM cytochalasin D that can abolish the TNT connections between PC12 cells. Our study shows that PS-exposed membrane delivered from to live cells through TNTs may act as signal for macrophages to induce phagocytosis of live cells.
Tumors are composed of different cell clones which show different susceptibility to drugs. However, little is known as to how drug-sensitive cells interact with drug-resistance cells during chemotherapeutic treatment. To investigate cell-to-cell communication under stress condition, we focused on chemotherapy induced bystander responses and TNTs connecting apoptotic cancer cells. 1. A microfluidic device as a tool for screening and optimization of putative chemotherapeutic drugs. By using the microfluidic approach we developed, targeted and bystander cell populations can be seed in two defined areas of the chip under uniform environmental conditions. This allows performing a spatio-temporal analysis of the drug-induced bystander effect. Furthermore, multiple channels can be integrated into a single chip which allows screening of a group of drugs simultaneous. Thus, our experimental approach used here to monitor bystander effects can be exploited for screening different cancer cells and drugs for optimized bystander effects and improved chemotherapies. 2. TNT is a potential therapeutic target in the treatment of disease We provide evidence for a link between the formation of microtubule-containing TNTs, the transfer of mitochondria and the presence of rescue effect in apoptotic stressed cells. This suggests a new recovery mechanism for injured cells that proliferate slowly or cannot regenerate, such as neurons and cardiomyocytes, which are prone to apoptosis after ischaemic attacks. On the other hand, the rescue effect may help drug-sensitive cancer cells to acquire survival signals from drug-insensitive cells and escape death during cancer treatment. In another study, we show that “eat-me” signals, such as phosphatidylserine, could transferred from apoptotic to live cells and induce the phagocytosis of live cells by macrophages. It is possible that the transfer of PS-membrane from dead drug-sensitive cells to drug-resist tumor cells may induce a bystander effect and suppress tumour growth during cancer treatment. Similarly, the transfer PS-membrane may also bring harmful effect to normal tissue that close to apoptotic cells. Therefore, TNTs may act as a double-edged sword in different pathological conditions and become a potential therapeutic target in the treatment of disease. Further studies are needed to find in vivo evidence and explore new therapeutic strategies targeting on microenvironment regulated by apoptotic cells.
Role of tunneling nanotubes in the interaction of cells during apoptosis
To study cell-cell interactions during apoptosis of cancer cells, we focused on tunneling nanotubes (TNTs) connecting cells. Our results revealed that (1) transfer of mitochondria via TNTs rescues apoptotic PC12 cells and (2) TNT-dependent transfer of phosphatidylserine membrane from apoptotic cells induces phagocytosis of connected healthy cells.
1 Formation and function of TNTs in cells under apoptotic stress It has been shown that intercellular exchange of mitochondria can rescue damaged cells. However, the rescue mechanism by how and when this event was accomplished remained unclear. In this study, we found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when co-cultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterised by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 (EB3) and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c, but did not enter into the execution phase of apoptosis characterised by caspase-3 activation. Moreover, mitochondria co-localised with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the co-culture. We conclude that TNTs-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context. 2 Role of PS-positive TNTs in phagocytosis The exposure of phosphatidylserine (PS) at the outer leaflet of the plasma membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequent phagocytosis by activated macrophages. We found that when apoptotic PC12 cells were co-cultured with healthy PC12 cells, they connected with neighbouring healthy cells by TNTs containing PS-exposed plasmamembrane. Live imaging shows that these TNTs can transport PS-exposed membrane to the healthy cells at the contact site of the TNTs. When murine RAW264.7 macrophages were added into the co-culture of apoptotic and healthy PC12 cells, there was an increase of phagocytosis of healthy PC12 cells comparing with co-cultures without apoptotic cells. Moreover, the phagocytosis of healthy cells was inhibited by low concentration of cytochalasin D which can abolish the TNT connections between cells. Thus, our study suggests that the transferred PS-membrane may act as target signal for macrophages to induce phagocytosis of healthy cells. This finding will improve our understanding of interaction of cells during apoptosis.
Formation and function of TNTs in cells under apoptotic stress
To investigate cell-cell interactions during apoptosis of cancer cells, we focused on the formation and function of tunneling nanotubes (TNTs) connecting apoptotic cancer cells. Our results indicate that (1) p53 is not the master gene for TNT formation and (2) a TNT-depdent recuse effect in apoptotice stressed cells.
1. Role of tumour suppressor protein p53 in TNT formation
TNTs are present in a broad range of cell types including primary cells and cancer cell lines. The discovery that TNT-like structures exist in vivo suggests they may play an important role during tissue development and maintenance. TNTs facilitate intercellular transfer of various cellular components and are thought to represent a novel form of cellular communication. p53 is an important tumour suppressor protein that regulates apoptosis and more than 50% of human tumors contain a mutation or deletion in the p53 gene. A recent study reported that p53 functions as a master gene for TNT formation in astrocytes. Therefore, we have investigated in collaboration with Prof. Bjørn Tore Gjertsen’s group (Haukeland University Hospital, Department of Internal Medicine) whether p53 is a key protein for the formation of TNTs.
Our data show that activation of p53 does not promote TNT formation in p53WT rat pheochromocytoma PC12 cells and OCI-AML3 (acute myeloid leukemia) cells. In addition, p53-negative human osteosarcoma cell lines (SAOS-2) and mesenchymal stromal stem cells (MSCs) from bone marrow of a double knock-out (dKO) (p53 -/- and mouse double minute 2 (MDM2) -/-)) C57BL/6 mouse can form TNTs. Exogeneous expression of p53 does not induce TNTs in SAOS-2 cells. In conclusion, our study revealed that p53 is not a master protein for TNT formation and further investigation is needed to dissect the molecular mechanisms underlying their formation.
2. Formation and function of TNTs in cells under apoptotic stress
Consistent with the model that TNTs are involved in cell-to-cell communication, apoptosis regulators may be transferred via TNTs between apoptotic and healthy cells and alter the fate of recipient cells. Indeed, TNTs were demonstrated to propagate death signals FasL between T lymphocytes and induce cell death. Also, TNTs may participate in the rescue of cardiomyoblasts from cell death by mesenchymal stem cells. However, the mechanism by which this transfer was accomplished under what stressed condition, remained unclear.
In this study, we show that apoptotic stimulated PC12 cells were rescued when co-cultured with healthy PC12 cells. Single cell analysis shows that stressed PC12 cells at early stages of apoptosis form a new type of TNT to interact with healthy, non-apoptotic cells. These TNTs have distinct cytoskeletal composition and biophysical properties when compared to TNTs interconnecting healthy cells. We observed that presence and transport of mitochondria in the TNTs. Importantly, when TNTs were eliminated by actin-depolymerizing drug or mitochondria of healthy cells were damaged, the rescue effect was inhibited. Our results suggest that the delivery of functional mitochondria via TNTs mediates the salvage of apoptotic cells.
Tamoxifen-induced bystander effects in breast cancer
Although evidence for the killing of cancer cells through drug-induced bystander effects exists, the underlying mechanism(s) remain unclear. By employing microfluidics, we were able to perform a spatial-temporal analysis of the bystander effect of tamoxifen, a chemotherapeutic drug frequently used in clinical treatment of breast cancer.
The bystander effect is the indirect inhibition or killing of tumor cells that are adjacent to those directly affected by radiation therapy or pharmacological treatments. In the case of radiation, identified mechanisms underlying bystander effects involve direct cell-cell communication via gap junctions or the release of factors such as cytokines, reactive oxygen species or nitric oxide. In the case of chemotherapy, much less is known about the involved mechanisms due to the difficulty to distinguish between target and bystander cells. This urges for new experimental approaches and strategies to overcome the limitations of currently used approaches, to unravel the spatial-temporal resolution and the molecular mechanisms of bystander effects.
Breast cancer therapy has been addressed in numerous studies as the most common invasive cancer for women. According to the molecular classification, more than 70% of breast tumors represent luminal subtypes. These are characterized by the presence of estrogen receptors and they are sensitive to anti-estrogen drugs. Furthermore, it has been shown that tamoxifen, an antagonist of the estrogen receptor, can reduce the occurrence of ER-positive tumors efficiently by 69%. Although the mechanisms underlying the tamoxifen-induced cell death in targeted cells have been extensively studied, it is unknown whether tamoxifen can produce bystander effects in breast cancer cells.
Here we studied the interaction of tamoxifen-treated human breast cancer MCF-7 cells with their neighboring counterparts by exploiting a microfluidic system for selective drug exposure to a subpopulation of cells. The spatio-temporal transfer of bystander responses from targeted to bystander cells was analyzed by measuring the mitochondrial membrane potential under conditions of free diffusion. Our data show that bystander effects were detectable as early as 1 hour after drug treatment and reached effective distances of at least 2.8 mm. Furthermore, under conditions precluding physical contact between targeted and bystander cells, we could show that the bystander effect was merely dependent on diffusible factors rather than cell contact-dependent signaling. Taken together, our study illustrates that the microfluidics approach is a promising tool to screen and optimize putative chemotherapeutic drugs for bystander effects.
Analysis of bystander effects during chemotherapy of cancer
Although evidence for the killing of cancer cells through drug-induced bystander effects exists, the underlying mechanism(s) remain unclear. By employing microfluidics, we were able to analyze the bystander effect of tamoxifen, a chemotherapeutical drug frequently used in clinical treatment, on breast cancer cell cultures directly by microscopy
The bystander effect is the indirect inhibition or killing of tumor cells that are adjacent to those directly affected by radiation therapy or pharmacological treatments. In the case of radiation, identified mechanisms underlying bystander effects involve direct cell-cell communication via gap junctions or the release of factors such as cytokines, reactive oxygen species or nitric oxide. In the case of chemotherapy, much less is known about the involved mechanisms due to the difficulty to distinguish between target and bystander cells. This results from the fact that both populations are exposed to the applied drugs. Further problems arise because (i) tumors are composed of different cell clones, which show diverse susceptibility to drugs, (ii) the knowledge as to how these clones interact with each other during chemotherapeutic treatment is limited, (iii) the crosstalk between tumor cells and their microenvironment, e.g. the attraction of fibroblasts by tumor tissue through adhesion molecules or tumor-derived soluble growth factors adds another level of complexity. For the latter case, fibroblasts are thought to promote tumor progression and cancer cell invasion through secretion of growth factors and ECM-degrading proteases. In conclusion, new experimental approaches and strategies are necessary to overcome the limitations of currently used approaches, to unravel the spatial-temporal resolution and the molecular mechanisms of bystander effects.
To get direct insights into bystander effects, we have started to employ microfluidics to analyze bystander responses between cancer cells. In contrast to the traditional practice of culturing cells, microfluidics permits (i) to distinguish between target and bystander cells by controlling their microenvironment and (ii) to discriminate between diffusible and cell contact-dependent cell-to-cell signaling. Furthermore, microfluidics provides an experimental model system to screen conditions and pharmaceutical compounds for their efficiency to produce bystander effects.
To establish the basic protocol for the bystander response analysis, breast cancer MCF-7 cells were seeded into a microfluidic chamber. Then one stripe of cells was pulse-treated with tamoxifen to induce apoptosis. Thereafter, the adjacent cell stripe, which was not treated with tamoxifen, was monitored for bystander effects with TMRM, a fluorescent indicator of apoptosis sensing the mitochondrial membrane potential. This revealed that not only the fluorescence of TMRM in tamoxifen-treated cells decreased but also non-treated cells showed a gradual loss of TMRM fluorescence indicative of bystander-induced apoptosis of cancer cells. This effect was observable already two hours after the pulse-treatment. Our future goal is to analyze if the observed bystander effect depends on soluble or cell-cell contact dependent signaling.
Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells.
Cell Death Differ 2015 Jul;22(7):1181-91. Epub 2015 jan 9
Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.
FASEB J 2015 Nov;29(11):4695-712. Epub 2015 jul 28
Tunneling nanotubes: Diversity in morphology and structure.
Commun Integr Biol 2014 Jan 1;7(1):e27934. Epub 2014 feb 6
Tunneling nanotube (TNT) formation is independent of p53 expression.
Cell Death Differ 2013 Aug;20(8):1124. Epub 2013 jun 14
Spatio-temporal analysis of tamoxifen-induced bystander effects in breast cancer cells using microfluidics
Biomicrofluidics 6, 024128 (2012)
Role of tunneling nanotubes in cell-to-cell communication
Lecture 08.11.2011, EMBL Heidelberg, Germany
Formation and function of tunneling nanotubes
Lecture 11.09.2011, EMBO Meeting, Vienna, Austria
Near-field studies on intercellular communication
- november 2012
- Hans-Hermann Gerdes